The role of the placenta is critical for fetal growth in ethanol abuse during pregnancy since it is the major organ supplying nutrients and oxygen to the fetus. Direct adverse effects of alcohol on placental cells could impair their functions and lead to restricted fetal growth and development. Impairment of placental blood flow in alcohol abuse during pregnancy has been suspected, but it is not known if this is due to alterations in vasodilator production or alterations in angiogenesis, the two components responsible for rises in placental perfusion during normal gestation. In addition, the timing of the ethanol exposure leading to fetal defects is unknown and there may be other confounders from other substances such as cocaine whose effects may obscure alcohol related abnormalities in the placenta. Recent confounders from other substances such as cocaine whose effects may obscure alcohol related abnormalities in the placenta. Recent studies in our laboratory have suggested that alcohol exposure may induce oxidative stress through generation of oxygen radicals that leads to higher levels of eNOS. NO release is decreased, we suspect, because more is required to remove the oxygen radicals and hence there is overall decreased stimulation of soluble guanylate cyclase resulting in the observed ethanol-induced decreased in tissue cGMP there is overall decreased stimulation of soluble guanylate cyclase resulting in the observed ethanol-induced decrease in tissue cGMP levels. This project proposes to study the mechanisms and significance of ethanol exposure in the human placenta with respect to the role of oxidative stress, the etiology of the decrease in NO and rise in eNOS and these collective changes on the angiogenic potential of the placenta. In addition to the perifused vi11i preparation, we will also utilize cultured trophoblasts and endothelial cells to study the compartments respectively. Consequently, the hypotheses to be tested are that: 1. Ethanol exposure in the human placenta alters blood flow regulatory mechanisms through the induction of oxidative stress. 2. Oxidative stress decreases NO bioavailability/production and increases eNOS production. 3. Both oxidative stress and decreased NO bioavailability/production stimulate the angiogenic potential of the human placenta is measured by mitogenesis and migration. We propose in turn to pursue the following specific aims: Specific Aim I: Define the relationship between ethanol exposure and oxidative stress through a comprehensive panel of oxidative stress markers in first and third trimester tissue and cells after short and long periods of exposure. Specific Aim 2: Define the relationship between ethanol exposure, oxidative stress, NO and eNOS levels in vi11i, trophoblasts and HUVEC-C by measuring NO release and eNOS expression after treatments with ethanol, NO substrate, NO inhibitor, inducers of oxidative stress and anti-oxidants. Ethanol exposure and eNOS functional activity through phosphorylation plus HUVEC-C NO release/NOS expression in response to shear stress will be examined. Specific Aim 3: Define the relationship between ethanol exposure, oxidative stress, NO levels, eNOS expression and angiogenic potential by measuring levels, and migration/mitogenic activity, or VEGF and bFGF in tissue, cells and conditioned media